DUGi

Aislamiento y caracterización de vesículas extracelulares del plasma seminal del burro

http://dugi.udg.edu/item/http:@@@@hdl.handle.net@@10256@@28660

Seminal plasma (SP) is a fluid made up of secretions produced in the accessory vesicles of the male reproductive system, specifically in the prostate and seminal vesicles. It has been shown that SP contains extracellular vesicles (EVs), which carry hormones, proteins, cytokines and other components that play a key role in the regulation of reproductive processes, influencing sperm function and the immune response in the female tract. The aim of this study was to isolate and characterise, for the first time, extracellular vesicles from donkey seminal plasma. Samples were collected from up to nine healthy, reproductively active donkeys. Samples were randomly pooled to create three groups (three SP samples per group). These groups were subjected to differential centrifugation and subsequently size exclusion chromatography (SEC) to separate two subsets of SVs: small (S-sEVs) and large (LsEVs). Orthogonal characterisation of EV samples was performed following MISEV 2023 guidelines, including morphology (by cryogenic electron microscopy), concentration (through measurement of total protein and total and CFSE-positive particles by flow cytometry), particle size distribution (by dynamic light scattering), purity (by albumin assessment by flow cytometry) and EV-specific protein markers (tetraspanins CD9, CD63 and CD81, and HSP70 by FC). The results indicated that donkey PS is highly enriched in EVs, showing a variation in size between the two subsets, with S-sEVs being smaller (~160 nm) and L-sEVs larger (~290 nm). Both subsets of EVs showed positive results for all four EV protein markers. However, the percentage of CD8l-positive events was higher in S-sEV samples compared to L-sEV samples (P<0.05). This study is the first to separate and characterise sEVs in donkey SP, highlighting their variability and suggesting differences in biogenesis and function between S-sEVs and L-sEVs

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